Development of the appropriate CD4+ T helper (TH) subset during an immune response is important for disease resolution. With the use of naïve, ovalbumin-specific alpha beta T cell receptor transgenic T cell, it was found that heat-killed Listeria monocytogenes induced TH1 development in vitro through macrophage production of interleukin-12 (IL-12). Moreover, inhibition of macrophage production of IL-12 may explain the ability of IL-10 to suppress TH1 development. Murine immune responses to L. monocytogenes in vivo are of the appropriate TH1 phenotype. Therefore, this regulatory pathway may have evolved to enable innate immune cells, through interactions with microbial pathogens, to direct development of specific immunity toward the appropriate TH phenotype.

We have examined the cells involved in the development of contact sensitivity to FITC in CBA mice. After skin painting with antigen, the number of dendritic cells (DC) in the draining lymph nodes increased by 30 min, was maximal at 48 h, and returned to normal by 6 d. Derivation of some DC from Langerhans' cells of the skin was indicated from the presence of Birbeck granules observed in some DC isolated 24 h after skin painting. The DC acquired FITC and by 8 h there were two populations, one highly fluorescent and the other less fluorescent. The highly fluorescent cells were present between 8 h and 3 d after sensitization, and during this period the DC were potent at initiating primary proliferative responses of normal syngeneic T lymphocytes in vitro. Between days 3 and 5 the numbers of lymphocytes in the draining lymph node increased. During this period purified T lymphocytes did not express detectable levels of antigen, but enriched B cell populations expressed antigen transiently on day 1, 2, or 3 after exposure to antigen. The results showed that, during a 3-d period after exposure to antigen, DC expressed antigen and stimulated T cell proliferation. We speculate that low amounts of FITC binding selectively to veiled cells or lymph node DC in the first hours after exposure to antigen are not immunogenic but that Langerhans' cells acquire high levels of antigen, enter the nodes, and initiate immune responses.

Summary A host's ability to resist certain pathogens such as Leishmania major can depend upon the phenotype of T helper (Th) subset that develops. Different murine genetic backgrounds are known to significantly alter the direction of Th subset development, although the cellular basis of this influence is poorly understood. To examine the basis of this effect we used an in vitro od3-T cell receptor (TCR) transgenic system for analysis of Th phenotype development. To control for TCR. usage, we derived the DOl1.10 c~/3-TCR transgene in several genetic backgrounds. Our findings suggest that the effects of genetic background on Th phenotype development reside within the T cell, and not the antigen-presenting cell compartment. Transgenic T cells from both the B10.D2 and BALB/c backgrounds showed development toward either the Thl or Th2 phenotype under the strong directing influence of interleukin (IL) 12 and IL-4, respectively. However, when T cells were activated in vitro under neutral conditions in which exogenous cytokines were not added, B10.D2-derived T cells acquired a significantly stronger Thl phenotype than T cells from the BALB/c background, correspondent with in vivo Th responses to Leishmania in these strains. Importantly, these cytokine differences resulted in distinct functional properties, because B10.D2-but not BALB/c-derived T cells could induce macrophage production of nitric oxide, an important antimicrobial factor. Thus, the genetically determined default Th phenotype development observed in vitro may correspond to in vivo Th subset responses for pathogens such as Leishmania which do not initiate strong Th phenotype-directing signals.

Sllmms.ryWe have previously shown that dendritic cells isolated after overnight culture, which can express B7 and are potent stimulators of naive T cell proliferation, are relatively poor at inducing the proliferation of a panel of murine T helper 1 (Thl) clones. Maximal stimulation of Thl clones was achieved using unseparated splenic antigen presenting cells (APC). An explanation for these findings is provided in the present study where we show that FcR + L cells transfected with B7 stimulate minimal proliferation of Thl clones in response to anti-CD3 antibodies, in contrast to induction of significant proliferation of naive T cells. However, addition of interleukin 12 (IL-12) to cultures of Thl cells stimulated with anti-CD3 and FcR + B7 transfectants resulted in a very pronounced increase in proliferation and interferon 3' (IFN-3") production. Exogenous IL-12 did not affect the B7-induced proliferation of naive T cells. This showed that whereas costimulatory signals delivered via B7-CD28 interaction are sufficient to induce significant proliferation of naive T cells activated through occupancy of the T cell receptor, Thl T cell clones require cooperative costimulation by B7 and IL-12. This costimulation was shown to be specific by inhibition of proliferation and IFN-y production using chimeric soluble cytolytic T lymphocyte-associated antigen 4-human IgG1Fc (CTLA4-Ig) and anti-IL-12 antibodies. Furthermore, the significant antigen specific proliferation and IFN-3" production by Thl clones observed when splenocytes were used as APC was almost completely abrogated using CTLA4-Ig and anti-IL-12 antibodies. Thus two costimulatory signals, B7 and IL-12, account for the ability of splenic APC to induce maximal stimulation of Thl clones. IL-10 downregulates the expression of IL-12 by IFN-3'-stimulated macrophages and this may account largely for the ability of IL-10 to inhibit APC function of splenic and macrophage APC for the induction of Thl cell proliferation and IFN-3' production. Indeed we show that IL-12 can overcome the inhibitory effect of IL-IO for the APC-dependent induction of proliferation and IFN-3" production by Thl clones. These results suggest that proliferation by terminally differentiated Thl clones, in contrast to naive T cells, requires stimulation via membrane-bound B7 and a cytokine, IL-12. It is possible that these signals may result in the activation of unresponsive T cells during an inflammatory response. IL-10, by its role in regulating such innate inflammatory responses, may thus help to maintain these T cells in an unresponsive state. M' embrane-bound structures on APCs provide costimulatory signals for the activation of T cell proliferation (1). The most dominant costimulatory interaction described to date is that between B7 and CD28 (2-7). CD28 is a homodimeric cell surface protein that is found on the majority of T lymphocytes (3,8,9). Its ligand, B7/BB1, is a membrane glycoprotein that is induced upon activation on B cells, macrophages (2, 10-13), and activated T cells (14) and is als...

We have examined the antigen presenting cell (APC) requirements for primary T cell activation and T helper (Th) cell phenotype differentiation using naive CD4+ T cells from alpha beta TCR transgenic mice. Purified dendritic cells were the principal cell required for induction of primary ovalbumin peptide specific T cell activation and clonal expansion. However, dendritic cells did not induce differentiation of T cells toward Th1 or Th2 phenotype. Addition of IL-4 during primary dendritic cell stimulations of T cells resulted in the development of a Th2 phenotype which produced high levels of IL-4 during secondary and tertiary stimulation. In contrast, development of Th1 cells producing high levels of IFN-gamma could not be induced with dendritic cells alone but required the addition of appropriately activated macrophages. Addition of splenic or peritoneal B cells did not induce Th1 development. Activated splenic macrophages induced Th1 development via a non-MHC restricted mechanism. Thus, requirements for induction of proliferation of naive CD4+ T cells are distinct from those directing Th1 phenotype development. IL-12 could replace the requirement for macrophages to induce Th1 development when T cells were activated with dendritic cells. Furthermore, this IL-12 mediated development of Th1 cells producing high levels of IFN-gamma was dependent on IFN-gamma.